Proteins present in cytoplasmic granules, including the pore-forming protein perforin, and a family of serine proteases called granzymes, have been implicated in granule mediated cytolysis. One of the proteins present in granules is the late T cell activation marker, granulysin. Granulysin is a 9 kDa arginine-rich protein, and is a member of a larger group of proteins, referred to as saposin-like proteins (sphingolipid activator protein like protein, SAPLIP). This family has been conserved for almost a billion years, from amoebas to humans. A subset of SAPLIP family members have antimicrobial activity. This subset includes NK-lysin, and amoebapores (which are used by amoebas to kill bacterial prey).
Granulysin is cytolytic against microbes and tumors. The crystal structure (see Anderson et al. (2003) J Mol. Biol. 325(2):355-65), suggests a mechanism for lysis of membranes. The five-helical bundle of granulysin resembles other “saposin folds”. Positive charges distribute in a ring around the granulysin molecule, and one face has net positive charge. Sulfate ions bind near the segment of the molecule identified as most membrane-lytic and of highest hydrophobic moment. The ion locations may indicate granulysin's orientation of initial approach towards the membrane. The crystal packing reveals one way to pack a sheet of granulysin molecules at the cell surface for a concerted lysis effort. The energy of binding granulysin charges to the bacterial membrane could drive the subsequent lytic processes. The loosely packed core facilitates a hinge or scissors motion towards exposure of hydrophobic surface that is proposed to tunnel the granulysin into the fracturing target membrane.
By electron microscopy, granulysin has been shown to trigger fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.
Experiments have been performed (see Ernst et al. (2000) J. Immunol. 165:7102-7108) to correlate the structure and function of granulysin using biophysical approaches. Synthetic peptides of granulysin conforming to a putative helix-loop-helix motif (aa 1-35, 36-70, and 31-50) were shown to retain 50-80% of anti-bacterial activity, whereas those peptides without this predicted structure (aa 1-20, 16-35, 46-65, 61-80) had <20% activity. The structural model also predicts that the α helices are amphipathic, including 15 positively charged amino acids: 12 arginine (16%) and three lysine residues. Chemical modification of the arginine residues caused complete inhibition of the antimicrobial effects of granulysin; however, modification of the lysine residues did not inhibit the antimicrobial activity. Granulysin altered bacterial membranes by increasing their permeability, inducing lesions on the surface of bacteria and separation of the cell wall and membranes from the cytoplasm. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on interactions with the microbial cell wall or membrane leading to increased permeability and osmotic lysis.
Since their introduction approximately 60 years ago, antibiotics have been our most powerful weapons against microbial invaders. However, the effectiveness of traditional antibiotics has been severely limited by the development of multi-drug resistant bacterial strains. In particular, analysis of clinical isolates of Propionibacterium acnes, a major etiologic agent of acne vulgaris, has indicated increasing resistance to standard antibiotic therapies, making the treatment of acne more challenging. Thus, there is a need for the development of new antimicrobial agents for the treatment of acne and other diseases with an infectious component.
Relevant Literature
U.S. Pat. No. 4,994,369 discloses the nucleotide and predicted amino acid sequence of the “519” protein, which was subsequently named granulysin. The sequences of granulysin variants may be accessed from the Genbank and EMBL databases, with the accession number X05044 for the mRNA sequence of 519; EMBL accession X05044 for the encoded protein; and EMBL:locus HSNKG5, accession X54101 for the NKG5 splice variant.
The sequence and structure of the granulysin gene is discussed in Jongstra et al. (1987) J. Exp. Med. 165:601-614; Donlon et al. (1990) Cytogenet. Cell Genet. 53:230-231; and Manning et al. (1992) J. Immunol. 148:4036-4042.
The use of granulysin as an antimicrobial agent may be found in U.S. Pat. No. 6,485,928, issued Nov. 26, 2002. The activity of granulysin and derivative peptides is discussed, for example, by Stenger et al. (1998) Science 282(5386):121-5; Wang et al. (2000) J. Immunol. 165(3):1486-90; and Kumar et al. (2001) Expert Opin Investig Drugs 10(2):321-9.